Application

Immunoblotting (see comments)

General description

Many membrane receptors for neurotransmitters and hormones are coupled to intracellular effectors and ion channels by heterotrimeric G-proteins. Receptor activation by ligand causes exchange, in a catalytic fashion, of GDP by GTP on the β-subunits of heterotrimeric G-proteins, followed by the dissociation of the β-subunit from the βγ-subunits. The latter reaction leads to regulation of a variety of effector systems. Recent evidence suggests that in addition to the β-subunit the βγ-subunits may also play a role in effector system regulation. The βγ-subunit has been implicated in the regulation of K⁺ channels, phospholipase C and A₂, and type II adenylylcyclase. A recent study demonstrated a role for βγ-subunits in targeting the β-adrenergic receptor kinase to membrane-bound receptors as well. This product is useful for quantification of β-subunit present in any particular cellular compartment and to study the role of G-protein in intracellular signaling. The purified standard is suitable for positive control or can be used to construct a standard curve using Anti-G-Protein, β-Subunit, Internal (127-139) RAbbit pAb (Cat. No. 371738).

Native G_βγ-subunit purified from bovine brain. Provides sufficient G-protein βγ-subunit for six standard curves. The βγ-subunit is biologically active and can be used in reconstitution experiments. Useful for quantitation of β-subunit present in any particular cellular compartment.

Legal Information

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Blank, J.L., et al. 1992. J. Biol. Chem.267, 23069.Bourne, H.R., et al. 1992. Cold Spring Harbor Symp. Quant. Biol.57, 145.Federman, A.D., et al. 1992. Nature356, 159.Lustig, K.D., et al. 1993. J. Biol. Chem.268, 13900.Pitcher, J.A., et al. 1992. Science257, 1264.Tang, W.J., and Gilman, A.G. 1992. Cell70, 869.

METHOD OF ASSAY:r>r>1. The stock solution contains 1250 ng of βγ in 25 µl. Prepare working standards from this stock solution as follows: Add 100 µl of solubilization buffer [20 mM Tris, 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 0.9% sodium cholate (pH 8.0)] and mix thoroughly. This provides a concentration of 10 ng/µl. Serially dilute with solubilization buffer to produce 5 ng/µl, 2.5 ng/µl, 1.25 ng/µl and 0.625 ng/µl. You should then have 62.5 µl of each of the standards except the 0.625 ng/µl which should be 125 µl. Complete preparation of the standards by adding 93.75 µl of sample buffer containing glycerol (25% w/v), tracking dye (0.05% w/v), SDS (5% w/v), glycine (3.6% w/v), and β-mercaptoethanol (10% v/v) to each standard except the 0.625 ng/µl, where you should add 187.5 µl to produce the desired concentration. r>A final standard containing the solubilization buffer and sample buffer in the ratio of 1:1.5 (10 µl solubilization buffer per 15 µl sample buffer) should also be prepared to give a zero standard. All standards should then be heated to 80°C for 4 min. The formulation described above provides 100, 50, 25, 12.5, and 6.25 ng of the βγ-subunit per 25 µl of volume. Alternative formulations are acceptable as long as these amounts of βγ-subunit are present for the preparation of a standard curve.r>2. The membrane or cellular organelle preparations should be solubilized (use the solubilization buffer described previously) on ice for 1 h. Thereafter, the preparation should be centrifuged to remove insoluble material; the amount of protein should be determined in the supernatant by an appropriate method. The solubilized protein should be diluted with solubilization buffer to obtain the desired amount of protein to be loaded in a volume of 10 µl. For each 10 µl of solubilized protein add 15 µl of the sample buffer. The preparations should be heated for 4 min at 80°C.r>3. Twenty-five (25) µl of each standard or sample should be loaded in each lane of a SDS-PAGE gel for electrophoresis by standard method. The resolved proteins should be transferred onto nitrocellulose or PVDF membranes by electrophoresis by standard methods. After blocking for 1 h with a standard blocking buffer [5% dried skim milk in buffer (pH 8.0) containing 50 mM Tris, 2 mM CaCl₂, 80 mM NaCl, 0.02% sodium azide, and 0.2% Nonidet P-40] the blot should be probed with the provided primary antibody diluted by a factor of 1:1000 in the blocking buffer for at least 1.5 h. After washing, the bands can be visualized by the method of your choice. Use iodinated goat anti-rabbit IgG when your intent is to construct a standard curve using densitometric readings from the blot.

Packaging

1250 ng in Plastic ampoule

Please refer to vial label for lot-specific concentration.

Physical form

In 50 mM HEPES, 1 mM DTT, 1 mM EDTA, 0.1% LUBROL™ Detergent, pH 7.6.

Reconstitution

Following initial thaw, aliquot and freeze (-70°C).

Warning

Toxicity: Standard Handling (A)